Composite

Part:BBa_K4452024:Design

Designed by: iGEM22_Hopkins   Group: iGEM22_Hopkins   (2022-09-30)


Ferritin with prSS4 transit peptide + RUBY + nptII


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 95
    Illegal BglII site found at 527
    Illegal BglII site found at 3059
    Illegal BglII site found at 7067
    Illegal BglII site found at 8151
    Illegal BamHI site found at 5716
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 3652
    Illegal NgoMIV site found at 4231
    Illegal NgoMIV site found at 5944
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 2370
    Illegal BsaI site found at 7462
    Illegal BsaI site found at 9397
    Illegal BsaI.rc site found at 2649
    Illegal BsaI.rc site found at 7741
    Illegal BsaI.rc site found at 9676


Design Notes

The three genes (BBa_K4452014, BBa_K4452017, BBa_K4452018) and a synthetic spacer (BBa_K4452009) were assembled into BBa_K4452024 using Golden Braid assembly, specifically level omega assembly with BsmBI.

Part Description BsmBI site
BBa_K4452014 Ferritin GGAG / AATG
BBa_K4452017 RUBY reporter AATG / AGCC
BBa_K4452018 nptII Antibiotic Resistance AGCC / TTCG
BBa_K4452009 M13R primer TTCG / CGCT
BBa_K4452010 pLX-B3(omega)1 GGAG / CGCT

GoldenBraid is a standardized assembly system based on type IIS restriction enzymes “that allows the indefinite growth of composite parts through the succession of iterative assembling steps.” This criteria is important for us because our cloning plan requires assembling basic parts into three distinct genes and then assembling those genes together. Additionally, GoldenBraid was designed with the intention of being an assembly standard for plant synthetic biology [1], so it would be an appropriate method for our project.

Implementation of GoldenBraid requires (1) specific type IIS restriction sites on basic parts and (2) specific destination plasmids with type IIS restriction sites positioned inside the vectors to allow for “braiding” parts binarily in indefinite successive iterations.

Basic parts are flanked with BsaI recognition-cleavage sites using distinct 4 bp cleavage sequences for neighboring basic parts such that the parts can be assembled in a specified sequence. When the parts are ligated with the correct destination plasmid that is flanked by BsaI sites in divergent orientation, all BsaI recognition sites disappear from the resulting expression plasmid. This process of putting the parts into a destination plasmid with BsaI digestion is referred to as level alpha assembly.

To assemble parts from level alpha plasmids into another destination plasmid requires BsmBI digestion, this is referred to as level omega assembly. The level alpha plasmids and the level omega destination plasmid will be flanked by complementary 4 bp BsmBI cleavage sites.

Source

none

References

[1] Sarrion-Perdigones A, Falconi EE, Zandalinas SI, Juárez P, Fernández-del-Carmen A, et al. (2011) GoldenBraid: An Iterative Cloning System for Standardized Assembly of Reusable Genetic Modules. PLOS ONE 6(7): e21622. https://doi.org/10.1371/journal.pone.0021622